Involvement of Thromboxane Receptor in the Proatherogenic Effect of Isoprostane F2-III
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《循环学杂志》
the Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia (M.T., Y.Y., L.V., D.P.), and Division of Cardiology, Washington University, St Louis, Mo (T.C.).
Abstract
Background— Atherosclerosis is a chronic inflammatory disease of the arterial wall, where it associates with oxidative stress and formation of oxidized lipids. The lipid oxidation product isoprostane iPF2-III, also known as 8-isoPGF2 and 15-F2t-IsoP, is elevated in patients with cardiovascular disease and present in atherosclerotic lesions. Several proatherogenic biological effects have been attributed to this isoprostane, suggesting that it could be an active factor in the pathogenesis of the disease.
Methods and Results— In this study we show that iPF2-III directly promotes atherogenesis in 2 different mouse models (ie, apolipoprotein E [apoE]– and LDL receptor–deficient mice) by activating the thromboxane receptor (TP). This effect is mediated by potent proinflammatory vascular reactions but is independent of thromboxane A2 levels, changes in blood pressure, or lipid profile. Pharmacological antagonism of TP suppresses the vascular proatherogenic effects of iPF2-III. Endothelial cells genetically lacking TP show reduced inflammatory responses when stimulated with this product of lipid oxidation but not other oxidized lipids.
Conclusions— Our results demonstrate that in atherosclerosis iPF2-III is not only a biomarker of oxidative stress but also an active mediator of its vascular phenotype. We conclude that in a clinical setting in which both thromboxane A2 and iPF2-III are elevated, suppression of the first alone would not provide the most benefit for patients because the coincidental presence of the isoprostane will still have a proatherogenic effect.
Key Words: atherosclerosis inflammation lipids pharmacology thromboxane
Introduction
Atherosclerosis is a chronic disease of the vasculature influenced by multiple exogenous and endogenous factors, which involves a complex interplay between certain blood components and the arterial wall.1 Inflammation plays an important role in atherogenesis and other cardiovascular diseases.2 On the other hand, oxidative stress is also widely recognized as a key factor in atherogenesis, where it is associated with vascular inflammatory reactions and formation of bioactive oxidized lipids.3 In recent years, the isoprostane iPF2-III, one of several isoprostanes produced in vivo, also known as 8-iso-PGF2, 8-epi-PGF2, or 15-F2t-IsoP, has emerged as a chemically stable product of lipid oxidation and a reliable marker of oxidative stress.4 Its formation is increased in animal models of atherosclerosis5 and in patients with atherosclerotic vascular diseases,6,7 patients with diabetes mellitus,8,9 and smokers.10 It is present in LDL undergoing oxidative modification and in human as well as animal atherosclerotic vascular lesions.11–13 A recent case-control study showed that a high circulating level of iPF2-III is an independent risk marker of coronary artery disease.14 Furthermore, its suppression coincides with reduced atherosclerosis in mice deficient for apolipoprotein E (apoE) and retarded progression of established vascular lesions in LDL receptor (LDLR)–deficient mice.13,15 Therefore, there is much associative evidence to support a role for this isoprostane in the development of atherosclerosis. Interestingly, iPF2-III possesses several biological activities, suggesting that theoretically it is not only a marker of lipid peroxidation but could also act as mediator of the vascular effects of oxidative stress. Thus, iPF2-III is a vasoconstrictor and affects platelets, smooth muscle cells, endothelial cells, and the interaction among these cell types, all of which have been implicated in atherogenesis.1,16
Clinical Perspective p 2874
In the present study we examine the functional role of iPF2-III in modulating the atherogenic vascular phenotype of 2 different mouse models, apoE-deficient and LDLR-deficient mice. Our data indicate that this product of lipid oxidation, but not others, promotes atherogenesis via a thromboxane receptor (TP)–mediated but thromboxane A2 (TXA2) level–independent mechanism.
Methods
Animals and Experimental Protocol
Mice were housed in a pathogen-free facility, and all of the animal procedures had the full approval of the University Institutional Animal Care and Use Committee. Unless otherwise stated, an equal number of male and female mice were used in each study. ApoE-deficient and LDLR-deficient mice, backcrossed 10 times to the C57BL/6 strain, were obtained from the Jackson Laboratory (Bar Harbor, Maine). At 10 weeks of age, mice were randomized in groups of 10 animals (5 males and 5 females) each to receive peritoneal injections of the different treatments 3 times per week. The first group received iPF2-III in PBS (1 μg/kg body wt); the second vehicle was also diluted in PBS. The final concentration of isoprostane selected was based on previous studies in which the compound has been shown to exert biological actions but not a significant acute effect on the vascular tone.4,17 A third group of mice was injected with another isoprostane, iPF3-III (1 μg/kg body wt), which does not possess vascular biological activity,4 and another group was treated with U46619 (1 μg/kg), a stable analogue of the endoperoxide PGH2 and a specific TP agonist.18 Finally, another group received iPF2-III (1 μg/kg) in association with a selective TP antagonist, SQ29548 (2 mg/kg),19 and another group received SQ28548 (2 mg/kg) alone. All the reagents were initially dissolved as stock solution in ethanol, and further dilutions were always made in PBS.
A separate group of apoE-deficient mice, while injected with iPF2-III (1 μg/kg), received aspirin in their drinking water (30 mg/L), which was replaced with fresh solution every other day. Previous studies have shown that this route of administration produces a cumulative effect in suppressing platelet cyclooxygenase-1 activity.20,21 The doses of the different compounds used in our experiments were selected on the basis of previously published studies.17–21
Another group of mice were injected intraperitoneally with [3H]iPF2-III (0.5 μCi) and then euthanized at 2 (n=4) and 24 hours (n=4). Thromboxane receptor–deficient (TP–/–) mice (a generous gift of Dr T.M. Coffman, Duke University, Durham, NC)22 and wild-type littermates (TP+/+) were injected with vehicle or the same amount of iPF2-III as described above. All the chemicals were from Cayman Chemical Co. The analyses described below were performed in a blinded fashion.
Biochemical Analyses
Plasma cholesterol and triglycerides were measured enzymatically, as previously described.13,15 Total plasma iPF2-III levels were measured by an enzyme immunoassay kit (Cayman Chemical Co); vitamin E levels were measured by a high-performance liquid chromatography assay method, as previously described.13,15 Urinary levels of 2,3-dinor-thromboxane B2 (TXB2) were measured as previously described.20 Circulating levels of soluble intercellular adhesion molecule 1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and endothelin-1 (ET-1) were measured by ELISA assays, as previously described.15 Serum paraoxonase 1 (PON1) activity was determined with the use of paraxon (O'-O-diethyl-O-p-nitrophenylphosphate; Sigma Chemical Co) as the substrate and measured by the increase in the absorbance at 412 nm due to the formation of 4-nitrophenol.23 Briefly, 15 μL serum was added to Tris/HCl buffer (100 mmol/L, pH 8.0) containing 1 mmol/L CaCl2 and 1 mmol/L paraoxon, and the generation of 4-nitrophenol was measured at 412 nm with an autoanalyzer. One unit of PON1 was defined as 1 nmol of 4-nitrophenol formed per minute under the aforementioned assay condition.
Blood Pressure Measurements
Systolic blood pressure and heart rates was measured 10 days before the end of the study with the use of a computerized, noninvasive, tail-cuff system, as previously described.21 One set of 10 measurements was obtained for each animal, and the mean blood pressure was calculated. Animals were habituated to the device before the blood pressures were measured to ensure accurate measurements.
Acquisition of Tissues
At the end of the injection period, mice were anesthetized, terminal blood samples were collected by puncture of the right ventricle, and mice were perfused with ice-cold PBS containing 20 μmol/L BHT and 2 mmol/L EDTA (pH 7.4), as previously described.21,24 Hearts were separated from the aorta at the base, embedded in OCT, and kept frozen at –20°C. After the removal of the surrounding adventitial fat tissue, the arch and thoracic aorta were opened longitudinally and fixed in formal/sucrose.
RNA Extraction and RNase Protection Assay
Abdominal aortas and liver were isolated and used to prepare RNA with the use of guanidium isothiocyanate, as previously described.24 Although aortas from 3 animals per group were pooled, liver from each animal was used for each RNA analysis. There was no effect of treatment on aortic weights or RNA yields. MCP-1, ICAM-1, ET-1, and GAPDH mRNA levels were assayed in the aorta, whereas PON1 and GAPDH mRNA levels were assayed in the liver. RNase protection assays were performed with 5 μg of total RNA with the use of a RiboQuant kit with a custom template set according to the manufacturer’s protocol (Pharmigen), as previously described.24 After RNase digestion, protected probes were resolved on denaturing polyacrylamide gels and quantified. The value of each hybridized probe was normalized to that of GAPDH included in each template set as internal control.
Quantification of Atherosclerotic Lesions
To evaluate the extent of atherosclerotic lesion formation, 2 vascular beds and 2 well-validated approaches were used, as previously described.13,15 The ascending aorta was used for cross-sectional microscopic analysis. Briefly, lesion size was determined from Oil Red O serial sections, cut 10 μm thick, and collected at 100-μm intervals, starting at the region where the aortic sinus becomes the ascending aorta. Lesion area was determined with the use of Image-Pro software (Media Cybernetic) on images that were captured with a color video camera connected with a dissection microscope, as previously described.13,15 Immunostaining for macrophage and smooth muscle cell content was performed in these sections. Briefly, a monoclonal antibody to mouse macrophage (MOMA-2; Accurate Chemicals) and an anti-human smooth muscle -actin monoclonal antibody (Sigma Chemical Co) were used, respectively. Antibody reactivity was detected with the use of a Nova red substrate kit (SK-4800, Vector Laboratory). Cross sections were counterstained with hematoxylin. As control, no primary antibody was added to the same sections.21,24
Next, the aortic arch together with the descending thoracic aorta was analyzed by the en face method. Briefly, after the removal of the surrounding adventitial fat tissue, the aorta was opened longitudinally, fixed in formal/sucrose, and stained with Sudan IV. The percentage of intimal surface area covered by atherosclerotic lesions was determined by capturing these images as described above.13,15
Statistical Analysis
Statistical analysis was performed with the program Graph Pad Prism 4.0. Results were expressed as mean±SD. ANOVAs that showed P<0.05 were followed with multiple pair comparison posttests with Bonferroni correction. Results from the studies with TP–/– and wild-type mice were also analyzed by a 2-way ANOVA with genotype, treatment, and their interaction used as the predictors.
Results
Effects of iPF2-III on Biochemical Measures and its Biodistribution
Ten-week-old apoE-deficient mice, while kept on regular chow diet, were randomized to the different group treatments, as described in Methods, for 8 weeks. By this time, we did not observe any difference in body weight, total plasma cholesterol, triglycerides, systolic blood pressure, and heart rate among the different groups (Table 1). Furthermore, no significant effect on LDL or HDL cholesterol concentrations was observed in any group (not shown). As expected, circulating plasma levels of total iPF2-III were significantly higher in mice receiving the active treatment than in those treated with vehicle (483±31 versus 285±23 pg/mL; P<0.01). To evaluate the biodistribution of the isoprostane used, we injected [3H]iPF2-III into a separate group of mice (n=8). After 2 and 24 hours, animals were euthanized; aorta, liver, kidney, and spleen were harvested, weighed, minced, and homogenized overnight, and tritium radioactivity was counted. Although we found that at 2 and 24 hours both liver and aorta had the highest accumulation, at the second time point the radioactivity was almost undetectable in the spleen and kidney (Table 2).
Effects of iPF2-III on Atherogenesis
At the end of the 8-week injection experiments, aortas were removed, and the size of the atherosclerotic vascular lesion was determined by 2 independent methods. Cross-sectional analysis of the proximal aorta showed that apoE-deficient mice treated with iPF2-III had significantly larger lesion area in the aortic valve compared with animals receiving vehicle (510±49 versus 981±100 μm2x103; P<0.01). By contrast, no effect was observed in the group of mice treated with iPF3-III (Figure 1). Similar findings were observed when the extension of the atherosclerotic lesion area was determined in the arch and thoracic aorta by the en face method. Thus, aortic lesion areas from mice receiving iPF2-III were significantly increased compared with those of control groups (Figure 1 and Figure 2). Because previous work showed that TP mediates some of the biological effects of this isoprostane, a fourth group of mice were randomized to receive iPF2-III (1 μg/kg body wt) plus SQ29548 (2 mg/kg body wt), and another group received the thromboxane mimetic U46619 (1 μg/kg body wt) alone or in combination with SQ29548 (2 mg/kg). Addition of SQ29548 completely prevented the proatherosclerotic effects of iPF2-III on the aortic tree (Figures 1 and 2). On the other hand, mice receiving U46619 showed an increase in atherosclerotic lesion area (1010±120 μm2x103), which, as expected, was prevented by the addition of SQ29548 (530±50 μm2x103; P<0.01). Confirming a previous report, we observed that SQ29548 alone also reduced aortic lesion area (not shown).25
Immunohistochemical analyses of aortic root sections showed no difference between vehicle- and iPF3-III–treated apoE-deficient mice in the percentage of area occupied by macrophages. However, mice receiving iPF2-III had a significant increase in this area, which was prevented by SQ29548 (Figure 1). No significant effect was observed for smooth muscle cells (not shown).
To further confirm the effect of this isoprostane on atherogenesis, we repeated the same experiments using the LDLR-deficient mice on chow. Ten-week-old mice were randomized to receive the same aforementioned treatments for the apoE-deficient mice. At the end of the study, cross-sectional analyses of the proximal aorta showed that only mice treated with iPF2-III had a significant increase in lesion area compared with controls (150±30 versus 457±65 μm2x103; n=6 per group; P<0.001), and this was prevented by SQ29548 (185±42 μm2x103; n=6). Similar to the apoE-deficient mice, no difference in lipid levels was observed among the 3 groups of mice (not shown).
Effects of iPF2-III on Platelet Activation
It is known that iPF2-III can activate platelets,4 which, in turn, by producing TXA2, could then contribute to its proatherogenic effect. For this reason, we evaluated whether treatment of the apoE-deficient mice with a dose of aspirin (30 mg/L), which may completely block platelet cyclooxygenase-1–dependent TXA2 biosynthesis,20,21 would prevent the vascular effects of iPF2-III. As predicted, at the end of the treatment urinary levels of 2,3-dinor-TXB2 were reduced by 95% in mice receiving aspirin (105±10 versus 12±3 ng/mg creatinine; P<0.001). However, as shown in Figure 1, aspirin did not influence the vascular response to iPF2-III, supporting a TXA2-independent mechanism for its proatherogenic effect.
Effects of iPF2-III on Vascular Inflammation
Because inflammation is a key factor in atherosclerosis and iPF2-III induces in vitro cellular inflammatory reactions, we next investigated the inflammatory vascular response to this treatment. After 2 weeks of treatment, circulating levels of MCP-1 and sICAM-1 were higher in mice treated with iPF2-III than in those receiving vehicle (215±20 versus 180±12 ng/mL and 16.9±1.1 versus 12±1.2 ng/mL; P<0.05 for both). At the end of the study, both cytokine levels were still significantly higher in mice treated with iPF2-III than in those receiving vehicle or iPF3-III. Although aspirin treatment did not modify the levels of both markers (not shown), the addition of SQ29548 to mice receiving iPF2-III significantly reduced both of them (Figure 3). By contrast, no change for plasma levels of ET-1 was observed among the different groups at any time point considered (not shown).
Next, we assayed the activity levels of serum PON1, an esterase associated with the HDL fraction in the plasma that has antioxidant and anti-inflammatory activities.26,27 Serum PON1 activity was unchanged between mice receiving vehicle or iPF3-III; however, this was significantly reduced after 2 weeks of treatment with iPF2-III (81±10 versus 58±11 U/mL). Lower levels of this marker were still observed at the end of the study, but they were normalized by SQ29548 (Table 1). By contrast, coincidental suppression of TXA2 levels by aspirin did not have any effect (not shown).
Endogenous levels of another antioxidant, vitamin E, were not significantly changed among the different groups of mice (Table 1). To further confirm these observations, we examined gene expression for all these inflammatory markers. RNase protection assay results revealed that treatment with iPF2-III significantly increased gene expression levels for MCP-1 and ICAM-1 but not ET-1 in the abdominal aorta but reduced PON1 mRNA levels in the liver of these mice compared with the mice treated with vehicle or iPF3-III (Figure 4). These changes were prevented by pretreatment with SQ29548 (Figure 4) but not aspirin (not shown).
Involvement of TP Activation in the Vascular Effects of iPF2-III
To further confirm the direct involvement of the TP in these biological effects, mice genetically deficient for the receptor (TP–/–) and their wild-type littermates (TP+/+) were injected following the same protocol. As shown in Table 3, we found that after 4 weeks of treatment, wild-type littermates (TP+/+) but not TP–/– mice injected with iPF2-III or U46619 had an increase in the levels of both MCP-1 and sICAM-1 but a decrease in PON1 activity compared with the vehicle control group. By contrast, no difference in plasma levels of ET-1 was found (Table 3). Similar results were also obtained when mRNA levels for those mediators were assayed in tissues form these 2 groups of mice (not shown). Finally, aortic rings isolated from a separate group of untreated TP–/– and wild-type littermate (TP+/+) mice (n=4 per group) were incubated for 24 hours with iPF2-III (10 μmol/L), 7-hydroxycholesterol (10 μmol/L), or 25-hydroxycholesterol (10 μmol/L), 2 lipids derived from the oxidation of cholesterol and implicated in atherogenesis,28 and 9-hydroxyoctadecaenoic acid (9-HODE) (10 μmol/L) or 13-HODE (10 μmol/L), lipids derived from the oxidation of linoleic acid. Supernatants from wild-type animals (TP+/+) showed a significant increase in both MCP-1 and ICAM-1 levels with all stimuli used. By contrast, whereas both oxysterols and hydroxy acids induced a somewhat similar increase in both cytokines in the absence of TP, incubation with iPF2-III did not have any effect (Figure 5).
Discussion
Atherosclerosis is a multifactorial chronic disease, which involves a continuous interplay between the arterial wall and components of the bloodstream.1 Consistent evidence has shown that vascular oxidative stress is a key factor in atherogenesis, where it is associated with inflammatory reactions and formation of oxidized lipids.1–3 Thus, much attention has been focused on specific oxidized lipid molecules, which are biologically active and derive from the oxidation of LDL or membrane phospholipids. Among them, the isoprostane iPF2-III, product of the free radical–mediated oxidation of arachidonic acid esterified to lipids, has emerged as a specific and sensitive marker of lipid peroxidation in vivo.29
Increased levels of this isoprostane are present in human and experimental atherosclerosis, and reduction of its levels is coincidental with decreased atherogenesis, suggesting a possible role for this oxidized lipid in the development of the disease.13,15 Interestingly, iPF2-III also possesses several biological activities in vitro, all of which are relevant to atherogenesis.4 Despite this body of evidence, an in vivo and more direct role for this isoprostane in the pathogenesis of atherosclerosis has not yet been provided. Our data establish that this chemically stable lipid oxidation product accelerates the development of atherosclerosis in 2 distinct mouse models, apoE- and LDLR-deficient mice. Its effect is directly mediated by the activation of the TP and subsequent induction of inflammatory vascular responses, but it is independent of de novo TXA2 biosynthesis or changes in plasma lipid levels.
Despite the fact that iPF2-III is known to be a vasoconstrictor, surprisingly, in our study we did not observe any significant effect on systolic blood pressure. This absence of effect could be attributed to the route by which we administered the compound, ie, intraperitoneally, whereas all the studies showing an effect on vascular tone have directly infused the compound into the vascular bed.30,31
We provided several lines of evidence in support of the involvement of the TP receptor in the in vivo proatherogenic vascular actions of the isoprostane F2-III: selective TP antagonism prevented them; a pure TP agonist reproduced them; and genetic deletion of TP abolished them.
Current views regard atherosclerosis as a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation.1 Inflammatory mediators appear to play a fundamental role in the initiation and progression of this chronic disease.3 Interestingly, in our study we found that the proatherogenic effect of iPF2-III is secondary to the induction of specific inflammatory mediators, such as sICAM-1 and MCP-1 but not ET-1, at the site of lesion development. By using pharmacological and genetic approaches, we provided evidence in support of the hypothesis that those effects are indeed mediated via TP activation. Importantly, from our results we can exclude that the upregulation of those proinflammatory factors is a mere consequence of the increased atherosclerosis for 2 main reasons. First, we observed an increase at an early time point (2 weeks of treatment) before the development of marked differences in lesion size or composition. Second, we observed a similar increase in wild-type littermates (TP+/+) but not in TP–/– mice.
Moreover, the proinflammatory response was associated with a significant early reduction of PON1 activity, an important endogenous anti-inflammatory system, which also has been implicated in atherogenesis.26,27 Once again, pharmacological blockade and genetic deletion of the TP prevented this effect. Although some data show a direct in vitro effect of iPF2-III on cytokine formation, the mechanism by which this isoprostane decreases PON1 activity is not clear currently, but some possibilities may be suggested. First, it could bind directly to plasma HDL and thus directly inhibit PON1. Second, by stimulating the secretion of inflammatory cytokines, it could activate phagocyte respiratory burst and induce generation of free radicals, which would ultimately downregulate PON1 gene expression. On the basis of the biodistribution experiments, we hypothesize that in our model iPF2-III accumulates in the vasculature and the liver, where it would then stimulate an inflammatory reaction, which ultimately results in the proatherogenic effect.
Over the past few years, it has become increasingly clear that iPF2-III is a sensitive marker of lipid peroxidation closely associated with the development of atherosclerosis. However, only indirect evidence has been provided to support a functional role for it in the vascular response to oxidative stress in vivo. In general, the link between this isoprostane and atherosclerosis was always thought to be that of a biomarker versus a mediator of atherosclerosis. Our present study revisits this dogma with the novel observation that this biologically active product of lipid oxidation, but not others, directly promotes atherosclerosis in 2 different animal models independently of TXA2 levels but via a specific TP-mediated mechanism, which involves the induction of potent vascular proinflammatory responses.
It is important to note some limitations of our study. One limitation is the use of animal models of atherosclerosis, which present significant differences from human disease even though they are well-controlled systems to test hypotheses. For instance, the disease process in the animal models is accelerated (months) compared with humans (decades). As a result, it is very difficult to know the extent to which studies like ours performed in mice can be extrapolated directly to humans. A second limitation is the fact that iPF2-III is not the most abundant or the most potent isoprostane in vivo. A third limitation is the dose of iPF2-III used in our experiments, which is higher than the concentration found in vivo in both animals and humans.29 Nonetheless, our results may have important clinical implications. Thus, in atherosclerosis, in which TXA2 and iPF2-III are both elevated, the suppression of the first alone would not provide the most benefit for patients because the coincidental presence of the isoprostane could still activate the TP, inducing a proatherogenic vascular phenotype. Interestingly, reduction of F2-isoprostane formation by vitamin E in combination with the suppression of TXB2 biosynthesis has been shown to be more effective than the 2 approaches alone in experimental atherosclerosis.32
We conclude that selective pharmacological strategies aimed at preventing the activation of this receptor during chronic treatment with cycloxygenase-1 inhibitors (aspirin-like drugs) may be rationally evaluated in the progression and evolution of human atherosclerosis.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (AG-11542, AG-22512) and the CART Fund. We thank Dr T.M. Coffman (Duke University, Durham, NC) for the generous gift of the TP–/– mice and Tao Ding for excellent technical assistance in these studies.
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Cyrus T, Tang LX, Rokach J, FitzGerald GA, Praticò D. Lipid peroxidation and platelet activation in murine atherosclerosis. Circulation. 2001; 104: 1940–1945.(Myan Tang, PhD; Tillmann )
Abstract
Background— Atherosclerosis is a chronic inflammatory disease of the arterial wall, where it associates with oxidative stress and formation of oxidized lipids. The lipid oxidation product isoprostane iPF2-III, also known as 8-isoPGF2 and 15-F2t-IsoP, is elevated in patients with cardiovascular disease and present in atherosclerotic lesions. Several proatherogenic biological effects have been attributed to this isoprostane, suggesting that it could be an active factor in the pathogenesis of the disease.
Methods and Results— In this study we show that iPF2-III directly promotes atherogenesis in 2 different mouse models (ie, apolipoprotein E [apoE]– and LDL receptor–deficient mice) by activating the thromboxane receptor (TP). This effect is mediated by potent proinflammatory vascular reactions but is independent of thromboxane A2 levels, changes in blood pressure, or lipid profile. Pharmacological antagonism of TP suppresses the vascular proatherogenic effects of iPF2-III. Endothelial cells genetically lacking TP show reduced inflammatory responses when stimulated with this product of lipid oxidation but not other oxidized lipids.
Conclusions— Our results demonstrate that in atherosclerosis iPF2-III is not only a biomarker of oxidative stress but also an active mediator of its vascular phenotype. We conclude that in a clinical setting in which both thromboxane A2 and iPF2-III are elevated, suppression of the first alone would not provide the most benefit for patients because the coincidental presence of the isoprostane will still have a proatherogenic effect.
Key Words: atherosclerosis inflammation lipids pharmacology thromboxane
Introduction
Atherosclerosis is a chronic disease of the vasculature influenced by multiple exogenous and endogenous factors, which involves a complex interplay between certain blood components and the arterial wall.1 Inflammation plays an important role in atherogenesis and other cardiovascular diseases.2 On the other hand, oxidative stress is also widely recognized as a key factor in atherogenesis, where it is associated with vascular inflammatory reactions and formation of bioactive oxidized lipids.3 In recent years, the isoprostane iPF2-III, one of several isoprostanes produced in vivo, also known as 8-iso-PGF2, 8-epi-PGF2, or 15-F2t-IsoP, has emerged as a chemically stable product of lipid oxidation and a reliable marker of oxidative stress.4 Its formation is increased in animal models of atherosclerosis5 and in patients with atherosclerotic vascular diseases,6,7 patients with diabetes mellitus,8,9 and smokers.10 It is present in LDL undergoing oxidative modification and in human as well as animal atherosclerotic vascular lesions.11–13 A recent case-control study showed that a high circulating level of iPF2-III is an independent risk marker of coronary artery disease.14 Furthermore, its suppression coincides with reduced atherosclerosis in mice deficient for apolipoprotein E (apoE) and retarded progression of established vascular lesions in LDL receptor (LDLR)–deficient mice.13,15 Therefore, there is much associative evidence to support a role for this isoprostane in the development of atherosclerosis. Interestingly, iPF2-III possesses several biological activities, suggesting that theoretically it is not only a marker of lipid peroxidation but could also act as mediator of the vascular effects of oxidative stress. Thus, iPF2-III is a vasoconstrictor and affects platelets, smooth muscle cells, endothelial cells, and the interaction among these cell types, all of which have been implicated in atherogenesis.1,16
Clinical Perspective p 2874
In the present study we examine the functional role of iPF2-III in modulating the atherogenic vascular phenotype of 2 different mouse models, apoE-deficient and LDLR-deficient mice. Our data indicate that this product of lipid oxidation, but not others, promotes atherogenesis via a thromboxane receptor (TP)–mediated but thromboxane A2 (TXA2) level–independent mechanism.
Methods
Animals and Experimental Protocol
Mice were housed in a pathogen-free facility, and all of the animal procedures had the full approval of the University Institutional Animal Care and Use Committee. Unless otherwise stated, an equal number of male and female mice were used in each study. ApoE-deficient and LDLR-deficient mice, backcrossed 10 times to the C57BL/6 strain, were obtained from the Jackson Laboratory (Bar Harbor, Maine). At 10 weeks of age, mice were randomized in groups of 10 animals (5 males and 5 females) each to receive peritoneal injections of the different treatments 3 times per week. The first group received iPF2-III in PBS (1 μg/kg body wt); the second vehicle was also diluted in PBS. The final concentration of isoprostane selected was based on previous studies in which the compound has been shown to exert biological actions but not a significant acute effect on the vascular tone.4,17 A third group of mice was injected with another isoprostane, iPF3-III (1 μg/kg body wt), which does not possess vascular biological activity,4 and another group was treated with U46619 (1 μg/kg), a stable analogue of the endoperoxide PGH2 and a specific TP agonist.18 Finally, another group received iPF2-III (1 μg/kg) in association with a selective TP antagonist, SQ29548 (2 mg/kg),19 and another group received SQ28548 (2 mg/kg) alone. All the reagents were initially dissolved as stock solution in ethanol, and further dilutions were always made in PBS.
A separate group of apoE-deficient mice, while injected with iPF2-III (1 μg/kg), received aspirin in their drinking water (30 mg/L), which was replaced with fresh solution every other day. Previous studies have shown that this route of administration produces a cumulative effect in suppressing platelet cyclooxygenase-1 activity.20,21 The doses of the different compounds used in our experiments were selected on the basis of previously published studies.17–21
Another group of mice were injected intraperitoneally with [3H]iPF2-III (0.5 μCi) and then euthanized at 2 (n=4) and 24 hours (n=4). Thromboxane receptor–deficient (TP–/–) mice (a generous gift of Dr T.M. Coffman, Duke University, Durham, NC)22 and wild-type littermates (TP+/+) were injected with vehicle or the same amount of iPF2-III as described above. All the chemicals were from Cayman Chemical Co. The analyses described below were performed in a blinded fashion.
Biochemical Analyses
Plasma cholesterol and triglycerides were measured enzymatically, as previously described.13,15 Total plasma iPF2-III levels were measured by an enzyme immunoassay kit (Cayman Chemical Co); vitamin E levels were measured by a high-performance liquid chromatography assay method, as previously described.13,15 Urinary levels of 2,3-dinor-thromboxane B2 (TXB2) were measured as previously described.20 Circulating levels of soluble intercellular adhesion molecule 1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and endothelin-1 (ET-1) were measured by ELISA assays, as previously described.15 Serum paraoxonase 1 (PON1) activity was determined with the use of paraxon (O'-O-diethyl-O-p-nitrophenylphosphate; Sigma Chemical Co) as the substrate and measured by the increase in the absorbance at 412 nm due to the formation of 4-nitrophenol.23 Briefly, 15 μL serum was added to Tris/HCl buffer (100 mmol/L, pH 8.0) containing 1 mmol/L CaCl2 and 1 mmol/L paraoxon, and the generation of 4-nitrophenol was measured at 412 nm with an autoanalyzer. One unit of PON1 was defined as 1 nmol of 4-nitrophenol formed per minute under the aforementioned assay condition.
Blood Pressure Measurements
Systolic blood pressure and heart rates was measured 10 days before the end of the study with the use of a computerized, noninvasive, tail-cuff system, as previously described.21 One set of 10 measurements was obtained for each animal, and the mean blood pressure was calculated. Animals were habituated to the device before the blood pressures were measured to ensure accurate measurements.
Acquisition of Tissues
At the end of the injection period, mice were anesthetized, terminal blood samples were collected by puncture of the right ventricle, and mice were perfused with ice-cold PBS containing 20 μmol/L BHT and 2 mmol/L EDTA (pH 7.4), as previously described.21,24 Hearts were separated from the aorta at the base, embedded in OCT, and kept frozen at –20°C. After the removal of the surrounding adventitial fat tissue, the arch and thoracic aorta were opened longitudinally and fixed in formal/sucrose.
RNA Extraction and RNase Protection Assay
Abdominal aortas and liver were isolated and used to prepare RNA with the use of guanidium isothiocyanate, as previously described.24 Although aortas from 3 animals per group were pooled, liver from each animal was used for each RNA analysis. There was no effect of treatment on aortic weights or RNA yields. MCP-1, ICAM-1, ET-1, and GAPDH mRNA levels were assayed in the aorta, whereas PON1 and GAPDH mRNA levels were assayed in the liver. RNase protection assays were performed with 5 μg of total RNA with the use of a RiboQuant kit with a custom template set according to the manufacturer’s protocol (Pharmigen), as previously described.24 After RNase digestion, protected probes were resolved on denaturing polyacrylamide gels and quantified. The value of each hybridized probe was normalized to that of GAPDH included in each template set as internal control.
Quantification of Atherosclerotic Lesions
To evaluate the extent of atherosclerotic lesion formation, 2 vascular beds and 2 well-validated approaches were used, as previously described.13,15 The ascending aorta was used for cross-sectional microscopic analysis. Briefly, lesion size was determined from Oil Red O serial sections, cut 10 μm thick, and collected at 100-μm intervals, starting at the region where the aortic sinus becomes the ascending aorta. Lesion area was determined with the use of Image-Pro software (Media Cybernetic) on images that were captured with a color video camera connected with a dissection microscope, as previously described.13,15 Immunostaining for macrophage and smooth muscle cell content was performed in these sections. Briefly, a monoclonal antibody to mouse macrophage (MOMA-2; Accurate Chemicals) and an anti-human smooth muscle -actin monoclonal antibody (Sigma Chemical Co) were used, respectively. Antibody reactivity was detected with the use of a Nova red substrate kit (SK-4800, Vector Laboratory). Cross sections were counterstained with hematoxylin. As control, no primary antibody was added to the same sections.21,24
Next, the aortic arch together with the descending thoracic aorta was analyzed by the en face method. Briefly, after the removal of the surrounding adventitial fat tissue, the aorta was opened longitudinally, fixed in formal/sucrose, and stained with Sudan IV. The percentage of intimal surface area covered by atherosclerotic lesions was determined by capturing these images as described above.13,15
Statistical Analysis
Statistical analysis was performed with the program Graph Pad Prism 4.0. Results were expressed as mean±SD. ANOVAs that showed P<0.05 were followed with multiple pair comparison posttests with Bonferroni correction. Results from the studies with TP–/– and wild-type mice were also analyzed by a 2-way ANOVA with genotype, treatment, and their interaction used as the predictors.
Results
Effects of iPF2-III on Biochemical Measures and its Biodistribution
Ten-week-old apoE-deficient mice, while kept on regular chow diet, were randomized to the different group treatments, as described in Methods, for 8 weeks. By this time, we did not observe any difference in body weight, total plasma cholesterol, triglycerides, systolic blood pressure, and heart rate among the different groups (Table 1). Furthermore, no significant effect on LDL or HDL cholesterol concentrations was observed in any group (not shown). As expected, circulating plasma levels of total iPF2-III were significantly higher in mice receiving the active treatment than in those treated with vehicle (483±31 versus 285±23 pg/mL; P<0.01). To evaluate the biodistribution of the isoprostane used, we injected [3H]iPF2-III into a separate group of mice (n=8). After 2 and 24 hours, animals were euthanized; aorta, liver, kidney, and spleen were harvested, weighed, minced, and homogenized overnight, and tritium radioactivity was counted. Although we found that at 2 and 24 hours both liver and aorta had the highest accumulation, at the second time point the radioactivity was almost undetectable in the spleen and kidney (Table 2).
Effects of iPF2-III on Atherogenesis
At the end of the 8-week injection experiments, aortas were removed, and the size of the atherosclerotic vascular lesion was determined by 2 independent methods. Cross-sectional analysis of the proximal aorta showed that apoE-deficient mice treated with iPF2-III had significantly larger lesion area in the aortic valve compared with animals receiving vehicle (510±49 versus 981±100 μm2x103; P<0.01). By contrast, no effect was observed in the group of mice treated with iPF3-III (Figure 1). Similar findings were observed when the extension of the atherosclerotic lesion area was determined in the arch and thoracic aorta by the en face method. Thus, aortic lesion areas from mice receiving iPF2-III were significantly increased compared with those of control groups (Figure 1 and Figure 2). Because previous work showed that TP mediates some of the biological effects of this isoprostane, a fourth group of mice were randomized to receive iPF2-III (1 μg/kg body wt) plus SQ29548 (2 mg/kg body wt), and another group received the thromboxane mimetic U46619 (1 μg/kg body wt) alone or in combination with SQ29548 (2 mg/kg). Addition of SQ29548 completely prevented the proatherosclerotic effects of iPF2-III on the aortic tree (Figures 1 and 2). On the other hand, mice receiving U46619 showed an increase in atherosclerotic lesion area (1010±120 μm2x103), which, as expected, was prevented by the addition of SQ29548 (530±50 μm2x103; P<0.01). Confirming a previous report, we observed that SQ29548 alone also reduced aortic lesion area (not shown).25
Immunohistochemical analyses of aortic root sections showed no difference between vehicle- and iPF3-III–treated apoE-deficient mice in the percentage of area occupied by macrophages. However, mice receiving iPF2-III had a significant increase in this area, which was prevented by SQ29548 (Figure 1). No significant effect was observed for smooth muscle cells (not shown).
To further confirm the effect of this isoprostane on atherogenesis, we repeated the same experiments using the LDLR-deficient mice on chow. Ten-week-old mice were randomized to receive the same aforementioned treatments for the apoE-deficient mice. At the end of the study, cross-sectional analyses of the proximal aorta showed that only mice treated with iPF2-III had a significant increase in lesion area compared with controls (150±30 versus 457±65 μm2x103; n=6 per group; P<0.001), and this was prevented by SQ29548 (185±42 μm2x103; n=6). Similar to the apoE-deficient mice, no difference in lipid levels was observed among the 3 groups of mice (not shown).
Effects of iPF2-III on Platelet Activation
It is known that iPF2-III can activate platelets,4 which, in turn, by producing TXA2, could then contribute to its proatherogenic effect. For this reason, we evaluated whether treatment of the apoE-deficient mice with a dose of aspirin (30 mg/L), which may completely block platelet cyclooxygenase-1–dependent TXA2 biosynthesis,20,21 would prevent the vascular effects of iPF2-III. As predicted, at the end of the treatment urinary levels of 2,3-dinor-TXB2 were reduced by 95% in mice receiving aspirin (105±10 versus 12±3 ng/mg creatinine; P<0.001). However, as shown in Figure 1, aspirin did not influence the vascular response to iPF2-III, supporting a TXA2-independent mechanism for its proatherogenic effect.
Effects of iPF2-III on Vascular Inflammation
Because inflammation is a key factor in atherosclerosis and iPF2-III induces in vitro cellular inflammatory reactions, we next investigated the inflammatory vascular response to this treatment. After 2 weeks of treatment, circulating levels of MCP-1 and sICAM-1 were higher in mice treated with iPF2-III than in those receiving vehicle (215±20 versus 180±12 ng/mL and 16.9±1.1 versus 12±1.2 ng/mL; P<0.05 for both). At the end of the study, both cytokine levels were still significantly higher in mice treated with iPF2-III than in those receiving vehicle or iPF3-III. Although aspirin treatment did not modify the levels of both markers (not shown), the addition of SQ29548 to mice receiving iPF2-III significantly reduced both of them (Figure 3). By contrast, no change for plasma levels of ET-1 was observed among the different groups at any time point considered (not shown).
Next, we assayed the activity levels of serum PON1, an esterase associated with the HDL fraction in the plasma that has antioxidant and anti-inflammatory activities.26,27 Serum PON1 activity was unchanged between mice receiving vehicle or iPF3-III; however, this was significantly reduced after 2 weeks of treatment with iPF2-III (81±10 versus 58±11 U/mL). Lower levels of this marker were still observed at the end of the study, but they were normalized by SQ29548 (Table 1). By contrast, coincidental suppression of TXA2 levels by aspirin did not have any effect (not shown).
Endogenous levels of another antioxidant, vitamin E, were not significantly changed among the different groups of mice (Table 1). To further confirm these observations, we examined gene expression for all these inflammatory markers. RNase protection assay results revealed that treatment with iPF2-III significantly increased gene expression levels for MCP-1 and ICAM-1 but not ET-1 in the abdominal aorta but reduced PON1 mRNA levels in the liver of these mice compared with the mice treated with vehicle or iPF3-III (Figure 4). These changes were prevented by pretreatment with SQ29548 (Figure 4) but not aspirin (not shown).
Involvement of TP Activation in the Vascular Effects of iPF2-III
To further confirm the direct involvement of the TP in these biological effects, mice genetically deficient for the receptor (TP–/–) and their wild-type littermates (TP+/+) were injected following the same protocol. As shown in Table 3, we found that after 4 weeks of treatment, wild-type littermates (TP+/+) but not TP–/– mice injected with iPF2-III or U46619 had an increase in the levels of both MCP-1 and sICAM-1 but a decrease in PON1 activity compared with the vehicle control group. By contrast, no difference in plasma levels of ET-1 was found (Table 3). Similar results were also obtained when mRNA levels for those mediators were assayed in tissues form these 2 groups of mice (not shown). Finally, aortic rings isolated from a separate group of untreated TP–/– and wild-type littermate (TP+/+) mice (n=4 per group) were incubated for 24 hours with iPF2-III (10 μmol/L), 7-hydroxycholesterol (10 μmol/L), or 25-hydroxycholesterol (10 μmol/L), 2 lipids derived from the oxidation of cholesterol and implicated in atherogenesis,28 and 9-hydroxyoctadecaenoic acid (9-HODE) (10 μmol/L) or 13-HODE (10 μmol/L), lipids derived from the oxidation of linoleic acid. Supernatants from wild-type animals (TP+/+) showed a significant increase in both MCP-1 and ICAM-1 levels with all stimuli used. By contrast, whereas both oxysterols and hydroxy acids induced a somewhat similar increase in both cytokines in the absence of TP, incubation with iPF2-III did not have any effect (Figure 5).
Discussion
Atherosclerosis is a multifactorial chronic disease, which involves a continuous interplay between the arterial wall and components of the bloodstream.1 Consistent evidence has shown that vascular oxidative stress is a key factor in atherogenesis, where it is associated with inflammatory reactions and formation of oxidized lipids.1–3 Thus, much attention has been focused on specific oxidized lipid molecules, which are biologically active and derive from the oxidation of LDL or membrane phospholipids. Among them, the isoprostane iPF2-III, product of the free radical–mediated oxidation of arachidonic acid esterified to lipids, has emerged as a specific and sensitive marker of lipid peroxidation in vivo.29
Increased levels of this isoprostane are present in human and experimental atherosclerosis, and reduction of its levels is coincidental with decreased atherogenesis, suggesting a possible role for this oxidized lipid in the development of the disease.13,15 Interestingly, iPF2-III also possesses several biological activities in vitro, all of which are relevant to atherogenesis.4 Despite this body of evidence, an in vivo and more direct role for this isoprostane in the pathogenesis of atherosclerosis has not yet been provided. Our data establish that this chemically stable lipid oxidation product accelerates the development of atherosclerosis in 2 distinct mouse models, apoE- and LDLR-deficient mice. Its effect is directly mediated by the activation of the TP and subsequent induction of inflammatory vascular responses, but it is independent of de novo TXA2 biosynthesis or changes in plasma lipid levels.
Despite the fact that iPF2-III is known to be a vasoconstrictor, surprisingly, in our study we did not observe any significant effect on systolic blood pressure. This absence of effect could be attributed to the route by which we administered the compound, ie, intraperitoneally, whereas all the studies showing an effect on vascular tone have directly infused the compound into the vascular bed.30,31
We provided several lines of evidence in support of the involvement of the TP receptor in the in vivo proatherogenic vascular actions of the isoprostane F2-III: selective TP antagonism prevented them; a pure TP agonist reproduced them; and genetic deletion of TP abolished them.
Current views regard atherosclerosis as a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation.1 Inflammatory mediators appear to play a fundamental role in the initiation and progression of this chronic disease.3 Interestingly, in our study we found that the proatherogenic effect of iPF2-III is secondary to the induction of specific inflammatory mediators, such as sICAM-1 and MCP-1 but not ET-1, at the site of lesion development. By using pharmacological and genetic approaches, we provided evidence in support of the hypothesis that those effects are indeed mediated via TP activation. Importantly, from our results we can exclude that the upregulation of those proinflammatory factors is a mere consequence of the increased atherosclerosis for 2 main reasons. First, we observed an increase at an early time point (2 weeks of treatment) before the development of marked differences in lesion size or composition. Second, we observed a similar increase in wild-type littermates (TP+/+) but not in TP–/– mice.
Moreover, the proinflammatory response was associated with a significant early reduction of PON1 activity, an important endogenous anti-inflammatory system, which also has been implicated in atherogenesis.26,27 Once again, pharmacological blockade and genetic deletion of the TP prevented this effect. Although some data show a direct in vitro effect of iPF2-III on cytokine formation, the mechanism by which this isoprostane decreases PON1 activity is not clear currently, but some possibilities may be suggested. First, it could bind directly to plasma HDL and thus directly inhibit PON1. Second, by stimulating the secretion of inflammatory cytokines, it could activate phagocyte respiratory burst and induce generation of free radicals, which would ultimately downregulate PON1 gene expression. On the basis of the biodistribution experiments, we hypothesize that in our model iPF2-III accumulates in the vasculature and the liver, where it would then stimulate an inflammatory reaction, which ultimately results in the proatherogenic effect.
Over the past few years, it has become increasingly clear that iPF2-III is a sensitive marker of lipid peroxidation closely associated with the development of atherosclerosis. However, only indirect evidence has been provided to support a functional role for it in the vascular response to oxidative stress in vivo. In general, the link between this isoprostane and atherosclerosis was always thought to be that of a biomarker versus a mediator of atherosclerosis. Our present study revisits this dogma with the novel observation that this biologically active product of lipid oxidation, but not others, directly promotes atherosclerosis in 2 different animal models independently of TXA2 levels but via a specific TP-mediated mechanism, which involves the induction of potent vascular proinflammatory responses.
It is important to note some limitations of our study. One limitation is the use of animal models of atherosclerosis, which present significant differences from human disease even though they are well-controlled systems to test hypotheses. For instance, the disease process in the animal models is accelerated (months) compared with humans (decades). As a result, it is very difficult to know the extent to which studies like ours performed in mice can be extrapolated directly to humans. A second limitation is the fact that iPF2-III is not the most abundant or the most potent isoprostane in vivo. A third limitation is the dose of iPF2-III used in our experiments, which is higher than the concentration found in vivo in both animals and humans.29 Nonetheless, our results may have important clinical implications. Thus, in atherosclerosis, in which TXA2 and iPF2-III are both elevated, the suppression of the first alone would not provide the most benefit for patients because the coincidental presence of the isoprostane could still activate the TP, inducing a proatherogenic vascular phenotype. Interestingly, reduction of F2-isoprostane formation by vitamin E in combination with the suppression of TXB2 biosynthesis has been shown to be more effective than the 2 approaches alone in experimental atherosclerosis.32
We conclude that selective pharmacological strategies aimed at preventing the activation of this receptor during chronic treatment with cycloxygenase-1 inhibitors (aspirin-like drugs) may be rationally evaluated in the progression and evolution of human atherosclerosis.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (AG-11542, AG-22512) and the CART Fund. We thank Dr T.M. Coffman (Duke University, Durham, NC) for the generous gift of the TP–/– mice and Tao Ding for excellent technical assistance in these studies.
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Cyrus T, Tang LX, Rokach J, FitzGerald GA, Praticò D. Lipid peroxidation and platelet activation in murine atherosclerosis. Circulation. 2001; 104: 1940–1945.(Myan Tang, PhD; Tillmann )